One of the most important developments in the 21st century is the use of RNAi to modulate expression in cells. Use of RNAi avoids the tedious use of knockout protocols, while allowing for the determination of the effect of reduced or eliminated expression of a protein on the phenotype of a cell. The major variations of RNAi are referred to as miRNA (microRNA), siRNA (short interfering RNA), shRNA (short hairpin RNA) and piRNA (piwi RNA).
For miRNA the conventional wisdom is that a gene encodes the miRNA in a form referred to as the primary-miRNA (pri-miRNA). The gene may be in any portion of the genome, frequently being found in regions that do not code for proteins and in introns. Not infrequently, a number of pri-miRNA genes are found in proximity, where the mature miRNAs will differ by only a few nucleotides, providing a group of isoforms that appear to have similar binding specificities and affinities. The expressed pri-miRNA will generally contain from a few hundred to a few thousand nucleotides. The pri-miRNA is then processed in the nucleus by the proteins Drosha and Pasha to the pre-miRNA that has a stem and loop structure with flanking sequences. The pre-miRNA will generally have about 60 to 70 bases. The pre-miRNA is then actively transported into the cytoplasm by exportin 5 and Ran-GTP. In the cytoplasm, the pre-miRNA is then further processed into small RNA duplexes of approximately 22 nucleotides by the proteins Dicer and Loquacious. The functional or guiding strand of the miRNA duplex is then loaded into the RNA-induced silencing complex (RISC). Finally, the miRNA guiding strand guides the RISC to the cognate messenger RNA (mRNA) target for translational repression or degradation of the mRNA. Mature miRNAs are thought to be the only functional species of miRNA genes that have direct role in target recognition. Pri- and pre-miRNAs are merely transitory intermediates during mature miRNA biogenesis and have no direct role in target recognition and repression.
The miRNA is frequently found to lack perfect complementarity with the target mRNA. Frequently, there are bulges, e.g. mismatches, deletions and insertions, not only between the target mRNA and the mature miRNA, but also between the two chains of the stem of the pre-miRNA. Also, it has frequently been found that more than one mRNA may be regulated by the same mature miRNA. A sequence of the miRNA from 5′-nucleotides 2-8. usually 2-7, is called the “seed” sequence. A sequence of from 7-8 nucleotides is found sufficient to recognize and bind to the target mRNA and provide translational repression or mRNA destabilization, while fewer nucleotides may still provide repression where there is substantial complementarity between the 3′ miRNA sequence and a target mRNA sequence in proximity to the sequence binding to the 5′ miRNA sequence.
There have been extensive efforts to define the sequences in the target mRNAs and the mature miRNAs that define effective binding between the two RNA species. It is frequently found that there are numerous mRNAs complementary to the same seed sequences, so that the miRNA has a potentially large repertoire for regulation. It remains a conundrum how the miRNA provides for specific regulation in light of the frequency of seed sequences and the substantial redundancy of seed sequences among miRNAs.
Because of the evident importance of miRNA in cell regulation—miRNAs have been found to be associated with cancers, cardiac and other diseases—there is great interest in understanding the mechanism whereby the miRNA regulates expression. In addition, the repertoire of miRNAs being expressed in a cell has been found to be associated with various indications and may indicate the severity of the indication and potentially a particular therapeutic protocol. Also, there is a great effort to develop miRNAs that may have therapeutic activity for the treatment of various diseases, such as cancer, where down regulation of one or more genes may inhibit tumor growth, control autoimmune diseases, correct genetic deficiencies associated with the expression of miRNAs and the like. In order to prepare arrays for screening miRNA profiles of cells it will be necessary to better understand the binding requirements between the miRNA and the target(s) mRNAs. To prepare drugs that have specificity for one or a few mRNAs, it will be essential to be able to design miRNAs that are specific for the desired targets and/or have substantially attenuated activity toward mRNAs other than the target mRNA. It will also be critical to be able to design antisense oligonuclotides (RNA and DNA) that can specifically silence miRNA genes that encode identical or nearly identical mature miRNAs. It is therefore of great interest to find additional components of the miRNA regulation that will allow improved identification of miRNAs and their targets, enhanced specificity for the miRNA toward the desired target and discrimination between the activities of miRNAs sharing substantial homology in the seed region.
Having the ability to specifically bind to one target mRNA provides a powerful tool for functional genomic screening. By being able to reduce expression of a specific protein one can determine the role the protein plays in the physiological processes of the cell. By employing RNAi using combinations of miRNA or its precursors, the interactions of the proteins and the effect on the physiology of the cell can be determined. The role of various proteins and combinations of proteins in native cells, cell lines, cancer cells, and the like can be investigated toward an understanding of the pathways of cells under various conditions.